Helicases are molecular motors that couple nucleotide binding and hydrolysis to nucleic acid unwinding and translocation. Many organisms encode multiple helicases that are essential to fundamental cellular functions such as DNA replication, repair, recombination, transcription and translation. Several human genetic disorders have also been linked to mutations in DNA helicases and helicases from human pathogen are being actively pursued as drug target. We propose to study the molecular mechanisms of two classes of helicases using the powerful single molecule fluorescence techniques that we and others have developed. Our approaches have already yielded several surprises and provided previously unattainable data on complex biochemical processes. For example, using single molecule fluorescence resonance energy transfer, we discovered that when a monomer of E. coli Rep helicases encounters a blockade during its 3'-5'translocation, it snaps back to the beginning site and repeats the same cycle multiple times, termed 'repetitive shuttling'. Further single molecule analysis suggested a plausible physical mechanism and biological implications of repetitive shuttling (Myong et al, Nature, 2005). On superfamily 1 helicases (Rep/UvrD/PcrA), we aim to (1) uncover the mechanisms of single stranded DNA translocation and repetitive shuttling, (2) determine the functional and regulatory roles of the observed large scale helicase conformation changes, and (3) visualize the coordination between helicase monomers during unwinding and measure the step sizes directly. (4) We will also study the mechanisms of two-tiered stepping of NS3, a superfamily 2 helicase from human pathogen Hepatitis C virus, and gain insight into the novel behaviors such as repetitive unwinding and gradual re-annealing. (5) We will also study how helicases function together with other proteins. (6) Finally, we will further develop our single molecule techniques for multicolor imaging and integration with microfluic devices. In all of our studies, bulk-phase biochemical experiments, crystallography, and computational studies through collaboration will help us design and interpret our measurements.